Amplification of the HER2 (ERBB2) gene occurs in approximately 25% of breast cancers and is associated with poor patient outcome. The antibody trastuzumab and the tyrosine kinase inhibitor lapatinib are the two approved drugs by the FDA for the treatment of HER2-positive breast cancer. Although clinically effective, many patients with HER2 gene-amplified breast cancer either do not respond or eventually escape trastuzumab and lapatinib, suggesting the presence of de novo and acquired mechanisms of drug resistance. In addition, molecular alterations involving the phosphatidylinositol-3 kinase (PI3K) signaling pathway are arguably the most frequent in breast cancer, encompassing together over 30% of invasive mammary tumors. In many cases, these alterations coexist with HER2 gene amplification and potentially mediate resistance to HER2 antagonists. We propose that, as a result of selective pressure by trastuzumab and/or lapatinib, breast cancers 1) upregulate compensatory survival signaling pathways which, in some cases, may be 'targetable' with an existing drug or combination of drugs, 2) utilize the PI3K/Akt pathway is a predominant mechanism of escape from anti-HER2 drugs which can be eliminated by concomitant use of PI3K inhibitors, and 3) 'acquire' or are enriched for mutations in the HER2 gene which may be present in a fraction of the HER2 alleles in tumors with HER2 gene amplification. To test these hypotheses, we propose the following specific aims: Aim 1. To determine the mechanism(s) by which mutations in PI3K enhance HER2-mediated transformation in human mammary epithelial cells. Aim 2. To determine whether genetic and/or pharmacological inhibitors of PI3K reverse resistance to HER2 inhibitors in HER2-overexpresing breast cancer cells with PIK3CA mutations. Aim 3. To determine molecular mechanisms of acquired resistance to the HER2 tyrosine kinase inhibitor lapatinib in HER2-overexpressing human breast cancer cells.